The thymus produces new T lymphocytes throughout life to maintain peripheral homeostasis and immune function. Unlike other tissues that undergo steady-state differentiation, the thymus contains no self-renewing stem or progenitor cells, and instead depends on constant recruitment of marrow-derived progenitors that circulate in the blood. The earliest intrathymic progenitors are multi-potent, but notably, lack B lineage potential, and thus do not correspond to any known progenitor in the marrow. Microenvironmental conditions inside the thymus induce these multipotent progenitors to adopt the T lineage fate, and to asymmetrically differentiate into multiple different T lineages. While Notch1 (N1) has been shown to play a key role in T lineage specification, it is inadequate to explain the complex process of generating multiple T lineages in the thymus. In conjunction with PHS award R21AI53739, we sought to identify other putative regulators of T lineage differentiation. Among the genes that we identified was Notch3 (N3). N3 knockout mice had already been generated and were found by other to be overtly normal, a finding that was confirmed in our laboratory. Surprisingly, however, we found that N3 deficiency results in a progressive, age-related degeneration of T progenitor activity in bone marrow. This phenotype is consistent with a human disease (CADASIL) associated with N3 deficiency (a heritable mutation), which is characterized by age-related (adult-onset) degeneration of vascular smooth muscle cells and recurrent strokes. Our current findings reveal N3 mutation to be the first known genetic defect leading to age-related degeneration of T lineage precursors in bone marrow. Further, the specificity of this requirement for N3 in marrow suggests that N3 may represent a marker for the long sought-after precursor to T lineage cells in marrow. Concisely stated, the goals of this project are 1) to expand and finalize for publication our finding that N3 is required to maintain T progenitor activity in marrow;2) to identify N3-expressing cells in marrow, and compare their lineage potentials to those of early intrathymic progenitors (notably, for the presence of B lineage potential);3) to ascertain whether N3 has a role in the thymus as well as the marrow, and to what extent this function overlaps with that of N1;and 4) to identify the targets of N3 signaling in the T lineage, and thus begin to define its molecular function. The approaches involve in vivo and in vitro assays for T lineage potential in bone marrow in mice at various ages;lineage tracing the progeny of marrow cells that signal through N3, using a N3:Cre fusion protein knock-in to conditionally activate a fluorescent reporter;in vivo and in vitro assessment of lineage potential in the reporter-positive cells;intrathymic deletion of N3, and intrathymic deletion of N1 at intermediate stage in N3-deficient mice;assessment of N3 signaling activity in thymus and marrow progenitors, using a N3:Gal3 fusion protein knock-in;and assessment of gene expression in cells that signal through N3, as well as their counterparts in young N3 knockout mice. T lymphocytes must be produced throughout life, a process that initiates with stem cells in the bone marrow, and is completed in the thymus. We have found that genetic mutation of a gene known as Notch3 results in an accelerated, age-related decline in the ability of the bone marrow to initiate T lymphocyte production by the thymus. Notch3 has also been implicated in another age-related degenerative disorder relating to strokes. This project is aimed at understanding how Notch3 functions to prevent senescence of the immune system, and may provide further insights into the role of Notch3 in strokes as well.